The specific aim of this project are designed to use spermatogonial transplantation to answer fundamental questions about testis physiology and spermatogenesis. The published method for spermatogonial transplantation involves the injection of crude germ cell preparations into recipient animals. Undifferentiated spermatogonial stem cells are believed to be the cell type that colonizes the seminiferous tubules of recipient mice and initiate spermatogenesis. We will inject an enriched population of spermatogonial stem cells from vitamin A deficient (VAD) mice into recipient testes to determine if this will enhance transplantation efficiency and if undifferentiated spermatogonia indeed colonize recipient testes. Additionally, germ cells from VAD mice and controls will be maintained in culture for various time periods prior to injection into recipient testes and transplantation evaluation. Spermatogonia from VAD mice and controls will be transfected and immediately injected into recipients to generate transgenic animals. Additionally, we will use germ cell culture to transfect spermatogonia prior to transplantation. Transfected germ cell transplantation and spermatogenesis in recipient mice would provide a novel and unique model to study germ cell and Sertoli cell gene expression during specific stages of spermatogenesis. In addition, this technique would provide an alternate method for the production of transgenic animals. Ultimately, these data will provide important information on the control of spermatogenesis and germ cell development. Furthermore, the potential application and significance of spermatogonial transplantation to the human clinical situation is enormous.